Ethanol currently has a huge market as an industrial solvent, and it is expected that ethanol can be actually used as a transportation fuel for automobiles and the like and the demand therefor continue to increase.
Glycerol (C3H8O3) is converted from glucose (C6H12O6) by one-step reduction and can provide improved reducing power during the metabolism of a microorganism. Many substances produced by fermentation frequently require reducing power in their metabolic pathways. Therefore, if glycerol can be effectively used as a substrate, the yield and productivity of the desired fermentation product can be improved. With a rapid increase in the production of biodiesel, the production of glycerol has increased, thus the price thereof is decreasing rapidly. As the production of biodiesel increases rapidly, the production of the byproduct glycerol also increases, and thus problems associated with the effective treatment of byproducts including glycerol will occur. Accordingly, the production of useful fermentation products by effective fermentation of glycerol will result in many effects.
The present inventors previously reported a transformant having an enhanced ability to produce ethanol from glycerol in Saccharomyces cerevisiae (Yu et al. Bioresour Technol. 101(11):4157-61 (2010)). In the studies of the present inventors, in order to increase ethanol productivity by improving a strain developed so as to use glycerol efficiently, an increase in glycerol productivity was achieved by blocking a pathway in which about 10% glycerol is produced as a byproduct in the production of ethanol in Saccharomyces cerevisiae. 
Strains known to be used in the microbial production of glycerol include yeasts such as S. cerevisiae, C. magnoliae, P. farinose, C. glycerinogens, bacteria such as B. subtilis, and algae such as D. tertiolecta. It is known that microorganisms generated by manipulating the glycerol biosynthesis pathway found in microorganisms known as glycerol-producing strains can be used. Generally, a carbon substrate such as glucose is converted to glucose-6-phosphate by hexokinase in the presence of ATP. Glucose-6-phosphate is converted by glucose-phosphate isomerase to fructose-6-phosphate which is then converted to fructose-1,6-diphosphate by 6-phosphofructokinase. The fructose-1,6-diphosphate is converted to dihydroxyacetone phosphate (DHAP) by aldolase. Finally, DHAP is converted to glycerol-3-phosphate (G3P) by NADH-dependent glycerol-3-phosphate dehydrogenase (G3PDH), and the G3P is then dephosphorylated to glycerol by glycerol-3-phosphate phosphatase (Hou J et al. Appl Microbiol Biotechnol. 85(4):1123-30 (2010)).
Among dehydrogenase genes that are involved in DHAP conversion to glycerol, GPD1 is known as a gene encoding glycerol-3-phosphate dehydrogenase that converts DHAP to glycerol-3-phosphate. In addition, GPP2 from Saccharomyces cerevisiae is known as a gene encoding glycerol-3-phosphate phosphatase that converts glycerol-3-phosphate to glycerol.
In the glycerol production pathway in Saccharomyces cerevisiae, dihydroxyacetone phosphate (DHAP) is converted by glycerol-3-phosphate dehydrogenase (GPD) to glycerol-3-phosphate, which is then converted by glycerol-3-phosphate phosphatase to glycerol which is then excreted from cells through the glycerol export channel Fps1 (Oliveira et al. Biochim Biophys Acta. 27; 1613(1-2):57-71 (2003)).
In order to increase ethanol production in a transformant constructed so as to use glycerol as a carbon source in Saccharomyces cerevisiae, two glycerol production genes, glycerol-3-phosphate dehydrogenase 2 and yeast glycerol channel Fps1, were deleted. In addition, it was found that glycerol uptake protein (Gup1) provides recovery against osmotic stress, and an increase in ethanol production was achieved.
Further, the present inventors developed broad regulatory functions of sigma factors for facilitating whole cell manipulation by TATA-binding proteins overexpression and induction of multiple simultaneous gene expression changes similarly.
Particularly in the case of ethanol production, the present invention can be applied to fungal cells and RNA polymerase II factors related to such eukaryotic cells. The present invention may encompass the use of other eukaryotic cells and transcriptional mechanisms corresponding to such cells, for improving phenotype characteristics, particularly the resistance of glycerol (and other sugars) and/or ethanol in culture medium and the production of ethanol by cells from various sources known in the art.
Thus, in the present invention, in order to increase ethanol production in a Saccharomyces cerevisiae engineered so as to use glycerol as a carbon source, a transformant that overexpresses TATA-binding proteins SPT3 and SPT15 was constructed, thereby increasing ethanol production.